Demonstration of IgE Antibodies to Nucleic Acid Antigens in Patients with Sle

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.

Development and characterization of monoclonal antibodies against WbSXP-1 for the detection of circulating filarial antigens

The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the World Health Organization. Presently, few immunodiagnostics are available for filarial monitoring programmes. The Wuchereria bancrofti (Wb) SXP-1 parasite protein, with 84% homology to Brugia malayi (Bm) SXP-1, was found to be highly immunogenic. WbSXP-1 is one among the diagnostic candidate molecules that were used for developing a rapid-antibody-flow-through diagnostic kit for filariasis. Studies were initiated with the aim of developing monoclonal antibodies against recombinant WbSXP-1 and prospective applications for the detection of both circulating Wb and Bm antigens in serum samples from infected individuals. The monoclones 1A6C2 of subclass IgG1k, and 2A12F8 of class IgM, specifically detected Wb and Bm microfilaria isolated from patients and did not show cross-reactivity with other filarial recombinant antigens. We anticipate that this work will address the problems faced in the rapid diagnosis of human lymphatic filariasis in endemic areas in developing countries.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

Mathematical Model of Viral Kinetics In Vitro Estimates the Number of E2-CD81 Complexes Necessary for Hepatitis C Virus Entry

Interaction between the hepatitis C virus (HCV) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells. The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally. Using the recently developed cell culture systems that allow persistent HCV infection in vitro, the dependence of HCV entry and kinetics on CD81 expression has been measured. We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry. Here, we constructed a mathematical model of HCV viral kinetics in vitro, in which we accounted explicitly for the dependence of HCV entry on CD81 expression. Model predictions of viral kinetics are in quantitative agreement with experimental observations. Specifically, our model predicts triphasic viral kinetics in vitro, where the first phase is characterized by cell proliferation, the second by the infection of susceptible cells and the third by the growth of cells refractory to infection. By fitting model predictions to the above data, we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells. We found that depending on the E2-CD81 binding affinity, between 1 and 13 E2-CD81 complexes are necessary for HCV entry. With this estimate, our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels, indicating that the estimate is robust. Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction. Further, our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection.

Functional regulation of PVBV Nuclear Inclusion protein-a protease activity upon interaction with Viral Protein genome-linked and phosphorylation

Regulation of NIa-Pro is crucial for polyprotein processing and hence, for successful infection of potyviruses. We have examined two novel mechanisms that could regulate NIa-Pro activity. Firstly, the influence of VPg domain on the proteolytic activity of NIa-Pro was investigated. It was shown that the turnover number of the protease increases when these two domains interact (as: two-fold; trans: seven-fold) with each other. Secondly, the protease activity of NIa-Pro could also be modulated by phosphorylation at Ser129. A mutation of this residue either to aspartate (phosphorylation-mimic) or alanine (phosphorylation-deficient) drastically reduces the protease activity. Based on these observations and molecular modeling studies, we propose that interaction with VPg as well as phosphorylation of Ser129 could relay a signal through Trp143 present at the protein surface to the active site pocket by subtle conformational changes, thus modulating protease activity of NIa-Pro. (C) 2011 Elsevier Inc. All rights reserved.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus

Background: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. Methods: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-gamma production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. Results: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-alpha and IFN-gamma) in T cells from non-human primates (NHPs) after BCG vaccination. Conclusions: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3 ` and 5 ` End Repair and Role of Polyprotein Processing in Viral Replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.

Viral Kinetics Suggests a Reconciliation of the Disparate Observations of the Modulation of Claudin-1 Expression on Cells Exposed to Hepatitis C Virus

The tight junction protein claudin-1 (CLDN1) is necessary for hepatitis C virus (HCV) entry into target cells. Recent studies have made disparate observations of the modulation of the expression of CLDN1 on cells following infection by HCV. In one study, the mean CLDN1 expression on cells exposed to HCV declined, whereas in another study HCV infected cells showed increased CLDN1 expression compared to uninfected cells. Consequently, the role of HCV in modulating CLDN1 expression, and hence the frequency of cellular superinfection, remains unclear. Here, we present a possible reconciliation of these disparate observations. We hypothesized that viral kinetics and not necessarily HCV-induced receptor modulation underlies these disparate observations. To test this hypothesis, we constructed a mathematical model of viral kinetics in vitro that mimicked the above experiments. Model predictions provided good fits to the observed evolution of the distribution of CLDN1 expression on cells following exposure to HCV. Cells with higher CLDN1 expression were preferentially infected and outgrown by cells with lower CLDN1 expression, resulting in a decline of the mean CLDN1 expression with time. At the same time, because the susceptibility of cells to infection increased with CLDN1 expression, infected cells tended to have higher CLDN1 expression on average than uninfected cells. Our study thus presents an explanation of the disparate observations of CLDN1 expression following HCV infection and points to the importance of considering viral kinetics in future studies of receptor expression on cells exposed to HCV.

Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity. (C) 2012 Elsevier Inc. All rights reserved.

Incentive Compatible Influence Maximization in Social Networks with Application to Viral Marketing

Information diffusion and influence maximization are important and extensively studied problems in social networks. Various models and algorithms have been proposed in the literature in the context of the influence maximization problem. A crucial assumption in all these studies is that the influence probabilities are known to the social planner. This assumption is unrealistic since the influence probabilities are usually private information of the individual agents and strategic agents may not reveal them truthfully. Moreover, the influence probabilities could vary significantly with the type of the information flowing in the network and the time at which the information is propagating in the network. In this paper, we use a mechanism design approach to elicit influence probabilities truthfully from the agents. Our main contribution is to design a scoring rule based mechanism in the context of the influencer-influencee model. In particular, we show the incentive compatibility of the mechanisms and propose a reverse weighted scoring rule based mechanism as an appropriate mechanism to use.

Microglia play a major role in direct viral-induced demyelination

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.